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PUBLISHED ONLINE: 10 MAY 2009 | DOI: 10.1038/NCHEM.207
Force-activated reactivity switch in a bimolecular
chemical reaction
Sergi Garcia-Manyes1 *, Jian Liang1, Robert Szoszkiewicz1, Tzu-Ling Kuo2 and Julio M. Fernández1 *
The effect of mechanical force on the free-energy surface that governs a chemical reaction is largely unknown. The
combination of protein engineering with single-molecule force-clamp spectroscopy allows us to study the influence of
mechanical force on the rate at which a protein disulfide bond is reduced by nucleophiles in a bimolecular substitution
reaction (SN2). We found that cleavage of a protein disulfide bond by hydroxide anions exhibits an abrupt reactivity
‘switch’ at 500 pN, after which the accelerating effect of force on the rate of an SN2 chemical reaction greatly
diminishes. We propose that an abrupt force-induced conformational change of the protein disulfide bond shifts its ground
state, drastically changing its reactivity in SN2 chemical reactions. Our experiments directly demonstrate the action of a
force-activated switch in the chemical reactivity of a single molecule.
T
he energy required for a chemical reaction to proceed is commonly provided by heat, light, electric potential or pressure1,2.
This energy allows the colliding molecules to overcome the
activation-energy barrier that governs the chemical reaction.
Mechanical force is a distinct, albeit less explored, way to activate
a chemical reaction because it can deform the reacting molecules
along a well-defined direction of the reaction coordinate1. Indeed,
activation of covalent bonds on the application of force to the reacting system by means of ultrasound was recently shown to change the
reaction pathways substantially, and leads to reaction products that
are different from those obtained when the reaction is initiated by
heat or light2. However, in these experiments the magnitude and
direction of the applied forces were not known. Furthermore, calculations of the density functions in the gas phase show how the application of relatively low forces can induce a substantial change in the
energy profile of a chemical reaction3.
At the single-molecule level, experiments conducted with atomic
force microscopy (AFM)4–6, supported by quantum-level simulations, measured the mechanical forces required for homolytic
and heterolytic bond rupture7. These studies show a continuous
deformation of covalent bonds along a one-dimensional Morselike potential with the application of a force6,8. In addition to
these observations, the vast single-molecule literature that has
accumulated over the past ten years demonstrates the ability of
mechanical forces to drive conformational changes in a diverse set
of molecules, including DNA, RNA, polysaccharides and proteins9–16. In spite of these advances, the relationship between
force-driven molecular conformations and complex chemical
reactions remains poorly understood.
A combination of protein-engineering techniques with singlemolecule force-clamp spectroscopy has made it possible to
monitor the reduction of individual disulfide bonds embedded
within a protein structure17,18. Our work at the single-bond level
directly demonstrated that the reduction of a protein disulfide
bond by a nucleophile is a force-dependent SN2 chemical reaction17.
These early experiments were carried out over a range of pulling
forces up to 500 pN, and showed that the rate of disulfide-bond
reduction increased exponentially with the pulling force, in excellent
agreement with a simple Arrhenius description of the energy barrier
1
that governs the reaction19. The main advantage of this experimental
approach is that it probes the transition state of the reaction with a
calibrated mechanical force and sub-ångström resolution19. For
example, we measured distances to the transition state (Dxr) of
the SN2 reaction that were in good agreement with quantum chemical calculations of the elongation experienced by the disulfide bond
at the transition state of the reaction19. In our early experiments, the
rate of disulfide-bond reduction by nucleophiles always followed a
simple exponential increase with the applied force, which marks a
single well-defined transition state for the reaction.
Here, we demonstrate that with hydroxide anions as the nucleophiles we can greatly extend the range of forces over which the SN2
reduction of disulfide bonds can be studied (to 2,000 pN).
Surprisingly, we found that the reduction of protein disulfide
bonds exhibits two distinct transition states that switch from
one to the other at a force of 500 pN. Our data show that at
low pulling forces (,500 pN) the reaction rate is limited by an
energy barrier with Dxr 0.5 Å, but at higher forces (.500 pN)
a different energy barrier dominates, with a much shorter Dxr ,
0.1 Å. Our results uncover a force-activated molecular switch
that greatly affects the reactivity of an SN2 chemical reaction. Our
experiments also illustrate the remarkable ability of force-clamp
spectroscopy to capture previously inaccessible details of the
energy profile of a protein-based chemical reaction.
Results
Using the experimental protocol of our previous studies, we engineered two point mutations (Gly32Cys and Ala75Cys) into the 27th
immunoglobulin-like domain of cardiac titin (I27), the mechanical
properties of which have been extensively characterized20. We constructed and expressed a polyprotein that consisted of eight identical
repeats of this modified domain, (I27G32C–A75C)8. In this construct,
each folded domain contains a single disulfide bond that is buried
and inaccessible to the external solvent environment. In our
force-clamp assay, we used a two-pulse force protocol to study the
force-dependent kinetics of the disulfide reduction17,21 (Fig. 1a).
The first force pulse (180 pN) rapidly unfolded all of the modules
in the polyprotein. Each unfolding event was marked by a step
increase in length of 11 nm, which corresponds to the extension
Department of Biological Sciences, 2 Department of Physics, Columbia University, New York, New York 10027, USA.
* e-mail: sergi@biology.columbia.edu; jfernandez@columbia.edu
236
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DOI: 10.1038/NCHEM.207
14 nm
F2
F1
11 nm
Cys 32
50 ms
OH−
Cys 75
2s
400 pN
F1
180 pN
F2
2s
14 nm
1s
1,000 pN
180 pN
550 pN
300 pN
1s
Figure 1 | Force-clamp spectroscopy monitors events in the reduction of single disulfide bonds with hydroxide anions in the solution. a, An AFM cantilever
picks up a polyprotein that contains eight repeats of the I27 module with disulfide bonds formed between cysteine residues engineered into positions 32 and
75. b, Using a double-pulse protocol (F1 and F2), we measured the rate at which the polyprotein disulfide bond was reduced by hydroxide anions. The first
force pulse (F1 ¼ 180 pN) unfolds and extends all the modules of the (I27G32C–A75C)8 polyprotein to the mechanical clamp created by the disulfide bonds,
which exposes them to the solution (blue line). Each unfolding event is marked by a step increase of 11 nm (inset, blue line, horizontal scale 50 ms).
A second, stronger force pulse tracks the time course of the disulfide-bond reduction, fingerprinted by equally spaced 14-nm steps that correspond to the
extension of the peptide chain released on the reduction of each disulfide bond (grey line). c, The rate of disulfide-bond reduction is accelerated by the
stretching force, as is apparent from the time course of disulfide reduction observed at 300 pN (red trajectory), F2 ¼ 550 pN (blue trajectory) and
F2 ¼ 1,000 pN (grey trajectory).
of the amino acids of the I27G32C–A75C protein up to the disulfide
bond. Unfolding exposes the buried disulfide bond to the solution,
which allows nucleophilic attack and thus reduction of the exposed
disulfide bonds (Fig. 1b, blue lines). Shortly after we had ensured the
unfolding of all the modules in the polyprotein, we applied second
force pulses of up to 2,000 pN to examine the force dependency of
the rate of reduction. In the presence of hydroxide anions
(0.25 mM, pH 11.4), the reduction of each disulfide bond is
marked by a 14 nm elongation step (Fig. 1b, grey line) that corresponds exactly to the total length of the residues trapped behind the
disulfide bond.
We varied the applied constant force of the second pulse and
measured the kinetics of disulfide reduction promoted by the
hydroxide anions. As is clear from Fig. 1c, the rate of reduction is
strongly influenced by force, rapidly increasing as the force is
increased from 300 pN (red trace) to 1,000 pN (grey trace). We
have not observed the 14 nm steps at neutral pH, at which the
concentration of hydroxide anions is vanishingly small
(1027 M). To obtain the rate of reduction at any particular force
or hydroxide anion concentration, we averaged and normalized
30–50 individual traces, such as those shown in Fig. 1, and fitted
the average trajectory to a single exponential (dotted lines in
Fig. 2a,b). From the exponential time constant of the fits (tR) we calculated the rate of reduction as r ¼ 1/tR. The standard error of the
mean (s.e.m.) of these data was estimated by bootstrapping. As
before, we fitted the force dependency of the rate of reduction
using the simple Arrhenius term r(F ) ¼ r(0)exp(FDxr/kT), where
r(0) is the rate constant in the absence of force17,19.
A semilogarithmic plot of the rate versus force measurements at
pH 11.4 and over an expanded force range up to 2,000 pN (Fig. 2c)
shows two clearly distinct regions marked by significantly different
exponential dependencies on the pulling force. It is clear that a
single Arrhenius term cannot fit the data of Fig. 2c. The first
regime of this plot, comprising data up to a force of 500 pN,
is described well by an Arrhenius term with Dxr ¼ 0.50 Å and
r(0) ¼ 8.2 1024 s21. However, fitting the Arrhenius term to the
data at forces higher than 500 pN revealed a novel conformation
of the transition state with a much shorter distance, Dxr ¼ 0.11 Å,
and r(0) ¼ 0.13 s21.
These experiments were repeated at different pH values to allow us
to measure the force-dependent reduction rate over a range of
hydroxide anion concentrations (Fig. 3a,b). As the pH is increased
(for example, pH . 13, Fig. 3a), the measured rate of reduction at
high forces approaches the upper rate limit of our spectrometer
(10–20 s21). Similarly, at lower pH values (11), the rate of reduction
measured at low forces is near the lower rate limit of the instrument
(0.05 s21). Nonetheless, the abrupt switch between force regimes of
the reaction is clear in all cases, at 500 pN (grey bar, Fig. 3a). The
rate–force curves obtained at different values of pH (Fig. 3a) are
simply shifted with respect to each other by the different
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1,200 pN
700 pN
pH 13.3
1.0
1.0
pH 12.3
500 pN
0.8
pH 11.4
0.8
480 pN
300 pN
0.4
pH 10.4
0.6
Pr (t)
0.6
Pr (t)
DOI: 10.1038/NCHEM.207
0.4
0.2
0.2
pH = 11.4
F = 500 pN
0.0
0.0
0
2
4
6
8
Time (s)
10
12
400
600
14
0
5
10
Time (s)
15
20
Rate (s−1)
10
1.0
0.1
800 1,000 1,200 1,400 1,600 1,800 2,000
Force (pN)
Figure 2 | The force dependency of the rate of disulfide-bond reduction by hydroxide anions exhibits two distinct reactivity regimes that switch at
500 pN. a, Measurement of the rate of disulfide-bond reduction at a constant pH of 11.4, at different pulling forces (range 300–1,200 pN). At each force we
averaged and normalized an ensemble of 30–50 traces obtained during the second force pulse (Fig. 1c) to obtain the probability of reduction, Pr.
The reduction rate at each pulling force is obtained by fitting a single exponential to the averaged trace (dotted black lines). b, Similar measurement of the
rate of reduction at a constant force of 500 pN, at different values of pH (range 10–13) obtained from Pr. c, Semilogarithmic plot of the disulfide-bond
reduction rate as a function of the stretching force. Error bars show the s.e.m. reduction rate obtained at each particular pulling force, estimated using
the bootstrapping method (see Methods). The reduction rate shows an abrupt change in force sensitivity at 500 pN. Fits of the Arrhenius term
r(F) ¼ r(0)exp(FDxr/kT) to the experimental data (green squares) up to 500 pN give Dxr ¼ 0.50 Å (dotted grey line). A much smaller distance
(Dxr ¼ 0.11 Å), which spans from 500 pN to 2 nN (dotted red line), is obtained when the experimental data are fitted to the high-force regime.
concentrations of hydroxide anions. We verified this by plotting the
rate of reduction measured at 500 pN against the hydroxide anion
concentration calculated from the pH of the solution (Fig. 3b).
The data show that the rate of reduction follows an approximately
first-order law that can be described as r(F ) ¼ k(F )[OH2]. Thus,
we can normalize the reduction-rate curves obtained at different
pH values (Fig. 3a) by dividing the measured rates r(F) by the concentration of hydroxide anions to give the reduction rate constant
k(F ). Plots of k(F ) versus force show that the experimental data
collapse into a single master curve, which clearly shows that the
two distinct force regimes coexist at 500 pN (Fig. 3c).
The results presented here unambiguously demonstrate that the
reduction of disulfide bonds by hydroxide anions exhibits an abrupt
change in reactivity at a force of 500 pN. However, is this a general
mechanism or a particular feature of nucleophilic attack by hydroxide anions? To answer this we measured the force dependency of the
rate of reduction with tris(2-carboxyethyl)phosphine (TCEP) as the
nucleophile19 over a wider force range up to 700 pN (crosses in
Fig. 3c). Our experiments rely on the non-specific attachment of
the protein between the surface and the cantilever tip. However,
the probability of detachment from any of the ends is exponentially
dependent on the stretching force. In the experiments with TCEP at
238
neutral pH, it proved impossible to obtain data at forces higher than
700 pN because the polyproteins picked up by the AFM cantilever
tip readily detached. The use of hydroxide anions in the measuring
solution greatly enhanced the attachment of the polyprotein to the
cantilever tip and the gold substrate, and thereby enabled exploration
of the reaction mechanisms under a much-expanded range of
pulling forces up to 2,000 pN, as demonstrated up to 1,400 pN in
Fig. 3c. Despite these difficulties, the TCEP data to 700 pN also
show an abrupt change in the slope at a force of 500 pN, which
demonstrates that the double-regime profile is not particular to
hydroxide anions. We also examined whether the specific location
of the disulfide bond within the structure of the I27 protein had a
role in the switch in reactivity at 500 pN. We repeated the experiments reported in Fig. 1 with two other polyprotein constructs,
(I27E24C–K55C)8 and (I27P28C–K54C)8 , in which the cysteine mutations
were engineered in different positions of the I27 sequence.
As before (Fig. 1), the reduction of a single, exposed disulfide
bond is identified by a step increase in length that corresponds
to the length of the amino acids trapped behind the disulfide
bond. Disulfide-bond reductions cause step increases in length of
10.4 nm for the (I27E24C–K55C)8 construct and of 8.8 nm for
the (I27P28C–K54C)8 construct (Supplementary Fig. S1).
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Rate (s−1)
10
1.0
pH 11.4
pH 12.3
pH 13.3
0.1
200
400
600
Force (pN)
800
1,000
20
1,000
k (M−1 s−1)
Rate (s−1)
15
10
100
pH 11.4
pH 12.3
pH 13.3
TCEP pH 7.4
10
5
F = 500 pN
1
0
0.00
0.05
0.10
0.15
0.20
[OH−] (M)
200
400
600
800
1,000
1,200
1,400
Force (pN)
Figure 3 | The reactivity switch observed at a 500 pN is a general mechanism in force-activated SN2 reactions. a, Force dependency of the disulfidebond reduction rate measured at three different values of pH (11.4, green squares; 12.3, grey triangles; 13.3, blue circles). Error bars show the s.e.m. reduction
rate obtained at each particular pulling force, estimated using the bootstrapping method (see Methods). b, The rate of disulfide-bond reduction measured at
500 pN is linearly dependent on the hydroxide anion concentration [OH2]. Thus, the reduction rate can be described by the equation r(F) ¼ k(F)[OH2],
where k(F) is the rate constant of the reaction. c, Semilogarithmic plot of the rate constant of reduction k(F) obtained by dividing the rates of reduction
shown in Fig. 3a by the hydroxide anion concentration. The data collapse to a single master curve that clearly shows two different sensitivities to the applied
force. The transition between the two regimes occurs in all cases at stretching forces 500 pN (grey bar). The force dependency of the rate of reduction of
disulfide bonds with the reducing agent TCEP (0.05–7.0 mM at pH ¼ 7.4, red crosses) also shows an abrupt change in reactivity at 500 pN, which
suggests that the reactivity switch is independent of the nucleophile.
Figure 4a,b shows the force dependency of the rate of reduction
for both constructs (Supplementary Fig. S2). Remarkably, the
two-regime profile with a transition point around 500 pN was also
found in the force dependency of the SN2 reaction for disulfide
bonds placed in different regions of the I27 protein. Fits of an
Arrhenius term to the experimental data obtained from the
(I27E24C–K55C)8 construct gave Dxr ¼ 0.46 Å and Dxr ¼ 0.07 Å for
the low- and high-force regimes, respectively (Fig. 4a). Fits to the
experimental data obtained from the (I27P28C–K54C)8 construct
gave Dxr ¼ 0.43 Å and Dxr ¼ 0.11 Å for the low- and high-force
regimes, respectively (Fig. 4b). These experiments were carried out
at a pH value that kept the rate of reduction to within the limits
of our spectrometer (see above). Interestingly, dividing the
reduction rate by the concentration of hydroxide anions for each
case resulted in reduction-rate constants k(F ) for all three polyprotein constructs that did not overlap (Fig. 4c). This demonstrates that
the chemical environment around each particular disulfide bond
has an important effect on the measured rate of reduction.
However, in all cases, the abrupt mode switch is observed at
500 pN (Fig. 4c, grey bar).
Discussion
Our single-molecule force-clamp assay directly measures fine details
of the transition state of protein-based chemical reactions. For
example, we measured Dxr with sub-ångström resolution, which
corresponds to the elongation of the S–S bond at the transitionstate structure. Our results show that at a critical force of
500 pN, the value of Dxr decreases abruptly, which marks a
drastic reduction in the sensitivity of the SN2 reaction to the applied
force. The vibrational frequency of a disulfide bond stretching
mode is 522 cm21, which predicts a bond-spring constant of
255 N m21 (ref. 22). Thus, a force of 500 pN would stretch this
bond by only 0.02 Å and, furthermore, this elongation would be
continuous with the applied force. Instead, we found an abrupt
change in the elongation of the disulfide bond at the transition
state, from Dxr 0.50 Å to Dxr 0.10 Å. We suggest two energy
scenarios that are consistent with these results.
The first considers that the force-accelerated SN2 chemical
reduction of the disulfide bond is described by an energy profile
composed of two consecutive energy barriers, an inner barrier
close to the reactants along the reaction coordinate, and an outer
barrier closer to the reaction products (Fig. 5a). In this scenario,
the inner barrier has a Dxinner of 0.1 Å and the outer barrier has a
higher activation-energy barrier with Dxouter ¼ 0.5 Å. The outer
barrier dominates the reaction in the low-force regime; however,
given that the outer barrier is more sensitive to force, its activation
energy (Ea) is more rapidly reduced until it is level with that of the
inner barrier, at 500 pN. Thus, at forces stronger than 500 pN
the inner barrier, far less sensitive to the stretching force, becomes
the rate-limiting step of the reaction (Fig. 5a). In this scenario, at
500 pN both activation-energy barriers have similar size. From
the extrapolation of the Arrhenius fit to the force dependency of
the disulfide reduction rate (Fig. 2c), we can calculate the reduction
rate in the absence of force, r(0), for both force regimes, before and
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10
Rate (s−1)
Rate (s−1)
10
DOI: 10.1038/NCHEM.207
1.0
1.0
I27E24C–K55C
I27P28C–K54C
0.1
0.1
400
600
800 1,000 1,200 1,400 1,600 1,800 2,000
Force (pN)
400
600
800 1,000 1,200 1,400 1,600 1,800 2,000
Force (pN)
105
k (M−1 s−1)
104
103
102
I27E24C–K55C
I27G32C–A75C
I27P28C–K54C
101
400
600
800
1,000
1,200
1,400
1,600
1,800
2,000
Force (pN)
Figure 4 | The reactivity switch is independent of the location of the disulfide bond in the protein structure. a, Semilogarithmic plot of the reduction rate
as a function of the pulling force for the (I27E24C–K55C)8 construct (at pH 12.3). The Arrhenius fit to the experimental data in the low-force regime (dotted
blue line) gives Dxr ¼ 0.46 Å, whereas the fit to the high-force regime (dotted grey line) gives Dxr ¼ 0.07 Å. b, Semilogarithmic plot of the reduction rate as
a function of the pulling force for the (I27P28C–K54C)8 construct (at pH 10.4). The Arrhenius fit to the experimental data in the low-force regime (dotted blue
line) gives Dxr ¼ 0.43 Å, whereas the fit to the high-force regime (dotted grey line) gives Dxr ¼ 0.11 Å. c, Normalization of the data shown in Figs 2c, 4a and
4b by the hydroxide anion concentration results in an effective rate constant that is dependent on the location of the disulfide bond within the structure of
the I27 protein. However, in all cases the reactivity switch is observed at 500 pN (grey bar).
after the 500 pN transition. Given that r(0) ¼ Aexp(–Ea/kBT ),
where kB is the Boltzmann constant, we estimate the energy
barriers that govern the process to be Ea,outer ¼ 86.1 kJ mol21 and
Ea,inner ¼ 73.5 kJ mol21 (DEa ¼ 12.6 kJ mol21), assuming that the
same pre-exponential factor A ¼ 1012 M21 s21 applies in the two
distinct force regimes. A molecular interpretation of this model
implies that a nucleophile first collides with the disulfide bond to
form a weak reactant complex that elongates the disulfide bond by
0.1 Å. The formation of this complex requires desolvation of the
reactants, which might be the origin of the first energy barrier
(inner barrier in Fig. 5a). After forming the reactant complex, the
reaction proceeds through its main energy barrier (outer barrier
in Fig. 5a), which causes a further bond elongation of 0.4 Å at
the transition state of the reaction. This reaction scheme (Fig. 5a)
is in qualitative agreement with the theoretical predictions applied
to the particular case of an ion–molecule reaction in the condensed
phase with asynchronous desolvation and ion–molecule complexation23–25. However, this model is inconsistent with the accepted view
that a bimolecular nucleophilic displacement (SN2) in solution
occurs in a single, concerted step26,27.
An alternative and more plausible explanation of our experimental results is that a mechanical force above 500 pN causes an abrupt
conformational change in the substrate disulfide bond, which modifies its ground state. In this scenario there is a single activationenergy barrier for the SN2 reaction, at which the shift in the
ground state of the disulfide bond moves the reaction-energy
barrier to a position closer to the transition-state structure
240
(Fig. 5b). From this new bond geometry, acquisition of the
transition state causes a much shorter bond elongation of 0.1 Å,
which accounts for the smaller force dependency observed in our
experiments. A molecular interpretation of this view requires an
understanding at the quantum chemistry level of the effects of a
pulling force on a disulfide bond, which is unavailable at present.
However, it is interesting that quantum calculations show that the
CS–SC dihedral angle, x, for open-chain disulfides is 858
(refs 28, 29) and that changing x from 858 to near 08 (cis conformation) or 1808 (trans conformation) causes the disulfide bond
S–S to lengthen by about 0.07 Å (refs 30–32). We speculate that
when the force applied to the substrate disulfide bond reaches
500 pN, an abrupt conformational change drives the disulfide
bond to a trans conformation, which elongates the Ca–Ca distance
of the cysteine residues, and so reduces the overall energy of the
molecule (Fig. 5c).
As discussed above, a disulfide bond in the trans conformation is
longer than that in the cis conformation. It is possible that this
lengthening is larger for a protein disulfide bond, and thus brings
the ground state of the reaction closer to the transition state. It is
likely that the force-induced trans geometry (Fig. 5c) results in a
change of the degree of the disulfide bond exposure to the solution,
which affects the new nucleophile-substrate orientation required for
the ‘backside’ SN2 reaction to proceed33,34. Remarkably, the calculated energy difference between both conformers ranges from 7
to 29 kJ mol21 (refs 31,32,35), similar to the energy difference
between the two barriers obtained from our experimental results,
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F
TSouter
TS
12.6 kJ mol−1
12.6 kJ mol−1
Free energy
Free energy
TSinner
= 180°
= 84.9°
Cα
F
Δxr = 0.1 Å
Δxr = 0.5 Å
Cα
Cα
Cα
Δxr = 0.4 Å
Δxr = 0.5 Å
F
Figure 5 | Schematics of the two energy scenarios compatible with the experimental results. a, Energy profile of the SN2 reaction for the two consecutive
energy barriers. The outer energy barrier (TSouter), with Dxr 0.5 Å, dominates the reaction in the low-force regime, and the inner energy barrier (TSinner),
much less sensitive to the pulling force (Dxr 0.1 Å), becomes rate-limiting above 500 pN. b, Energy profile of an SN2 reaction limited by a single barrier.
According to this scenario, at a force of 500 pN the S–S bond undergoes a conformational change that shifts the ground state by 0.4 Å closer to the
transition state (arrow). From this new geometry, the S–S bond elongates only by 0.1 Å to reach the transition-state structure. c, Hypothetical model for
of a conformational change induced by a mechanical stretching force that alters elongates the Ca–Ca distance of the cysteine residues and the dihedral angle
x (green planes) of a disulfide bond from the equilibrium conformation (yellow spheres, x ¼ 84.98) to the trans conformation (x ¼ 1808).
DEa ¼ 12.6 kJ mol21. Abrupt force-driven conformational changes in
a single molecule are common, such as chair–boat conformational
changes in single polysaccharide molecules that take place at a welldefined force16,36. For example, amylose molecules undergo forcedriven chair–boat transitions at 275 pN, whereas dextran molecules
undergo these transitions at 850 pN (refs 16,36,37). Thus, it is
entirely plausible that a force of 500 pN triggers an abrupt change
in the dihedral angle of the substrate disulfide bond.
Although both scenarios discussed here provide plausible explanations of our experimental results, other reaction mechanisms are
worth considering. For example, it is plausible that, in addition to
the S–S bond cleavage, C–S bond rupture occurs. Scission of the
C–S bond is compatible with a bimolecular elimination mechanism
induced by hydroxide anions38. In this scenario, the C–S bond
breaks at low forces, and with increasing bond angles and dihedral
angles, the S–S bond becomes more fragile or is more easily attacked
by hydroxide anions. However, the observation that the reactivity
switch is present at the same stretching force when TCEP is used
as a nucleophile argues against this possibility. In addition, more
complex mechanisms that involve both hydroxide anions and
other molecules may also explain our data. For example, both
ends of the breaking bond could react simultaneously under the
effect of a pulling force7,39,40, so hydroxide anions may react with
one of the sulfur atoms, whereas the other sulfur atom binds to
H2O. This trimolecular reaction would help trigger the reaction at
low forces. However, as it requires a very ordered transition state,
it would not have a major role at high forces.
Single-molecule force-clamp spectroscopy is a novel experimental tool with which to study the sub-ångström changes in the transition-state structure of chemical reactions in the solution phase.
Our experimental results provide the first demonstration that the
application of a threshold mechanical force to the substrate of a
bimolecular chemical reaction causes an abrupt change in reactivity.
Although the existence of such a mechanochemical switch is clearly
demonstrated by our experimental results, its molecular origins are
still unknown. To establish the molecular mechanisms that underlie
chemical reactions under force requires the development of
quantum mechanical simulations modified to accept a mechanical
stretching force as the driving perturbation7,39–41. Their development
would positively impact our understanding on the mechanisms by
which mechanical forces modulate chemical reactions, a mechanism
that is widespread in nature.
Methods
Protein engineering. We used a cysteine-free I27 protein in which native Cys 47 and
Cys 63, which do not form a disulfide bond, were mutated to alanines. Such
constructs were the platform on which we engineered the I27 mutants used in this
study. Using the QuickChange site-directed mutagenesis kit (Stratagene), we
introduced additional mutations to form disulfide bonds at specific positions within
the I27 protein sequence: Gly32 to Cys, Ala75 to Cys (I27G32C–A75C); Pro28 to Cys,
Lys54 to Cys (I27P28C–K54C); Glu24 to Cys, Lys55 to Cys (I27E24C–K55C). We
constructed an eight-domain N–C linked polyprotein for each I27 mutant. Such
polyproteins were engineered by consecutive subcloning of the respective monomers
using the BamH I, Kpn I and Bgl II restriction sites. The eight-domain polyproteins
were cloned into the pQE80L (Qiagen) expression vector and transformed into the
BLR DE3 Escherichia coli expression strain. Each polyprotein construct was finally
purified by histidine metal-affinity chromatography with Talon resin (Clontech) and
by gel filtration using Superdex 200 HR column (GE BioSciences).
Force spectroscopy. Force-clamp AFM experiments were conducted at room
temperature using a home-made set-up under force-clamp conditions described
elsewhere42. The sample was prepared by depositing 1–10 ml of protein in PBS
solution (at a concentration of 1–10 mg ml21 for polyproteins) onto a freshly
evaporated gold coverslide. Each cantilever (Si3N4 Veeco MLCT-AUHW) was
individually calibrated using the equipartition theorem, which gave a typical spring
constant of 15 pN nm21. Single proteins were picked up from the surface by
pushing the cantilever onto the surface with a contact force of 500–1,000 pN to
promote the non-specific adhesion of the proteins on the cantilever surface. The
piezoelectric actuator was then retracted to produce a set deflection (force), which
was kept constant throughout the experiment using an external, active feedback
mechanism while the extension was recorded. The force feedback was based on a
proportional, integral and differential amplifier, the output of which was fed to the
piezoelectric positioner. The feedback response is limited to 3–5 ms. The highresolution piezoelectric actuator gave our measurements of protein length a peak-topeak resolution of 0.5 nm. Data from the force traces were filtered using a pole
Bessel filter at 1 kHz. Experiments with TCEP were conducted in a sodium
phosphate buffer solution, specifically 50 mM sodium phosphate (Na2HPO4 and
NaH2PO4), 150 mM NaCl, pH 7.4. Experiments at different pH values were
conducted using buffer solutions of N-cyclohexyl-3-aminopropanesulfonic acid for
pH 10–11.5 and phosphate buffer solution for pH 12–13.5. The final pH value in
each solution was adjusted by adding the required amounts of NaOH or HCl
solutions. Solutions were filtered through a 0.2-mm membrane prior to
each experiment.
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241
ARTICLES
NATURE CHEMISTRY
Data analysis. All data were recorded and analysed using custom software written in
Igor Pro 5.0 (Wavemetrics). The fingerprint of a single polyprotein in our
experiments was considered to be at least four well-resolved steps of the
corresponding height in each construct during the first force pulse. No traces that
included unfolding events during the second force pulse were included in the
analysis. To obtain the reduction rate at each particular pulling force, we summed
and normalized numerous (n ¼ 30–100) reduction recordings for each particular
pulling force. To obtain the reduction rate at each particular pulling force, we fitted
the resulting averaged traces with single exponentials. To estimate the error on our
experimentally obtained rate constants, we used the non-parametric bootstrap
method43. At a given value of force, n staircases were randomly drawn with
replacement from our original data set. These were summed and fitted to obtain a
rate constant. This procedure was repeated 500 times for each data set, which
resulted in a distribution that provided the s.e.m. reduction rate. The s.e.m. of r(F )
was used as the weighting factor when the r(F) data were fitted to extract the values
of Dxr in the Arrhenius equation.
Received 12 January 2009; accepted 2 April 2009;
published online 10 May 2009
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Acknowledgements
We thank L. Dougan, J. Alegre, P. Kosuri and other members of the Fernández laboratory
for critical reading of the manuscript. S.G.-M. thanks the Generalitat de Catalunya for a
postdoctoral fellowship through the NANO and Beatriu de Pinós programs, and also the
Fundación Caja Madrid for financial support. This work was supported by NIH Grants
(to J.M.F.).
Author contributions
S.G.M. and J.M.F. conceived and designed the experiments; S.G.M., J.L., R.S. and T.K.
performed the experiments; S.G.M., J.L., R.S. and T.K analysed the data; S.G.M. contributed
materials and analysis tools; S.G.M and J.M.F. wrote the paper.
Additional information
Supplementary information accompanies this paper at www.nature.com/naturechemistry.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/. Correspondence and requests for materials should be addressed to
S.G.M. and J.M.F.
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© 2009 Macmillan Publishers Limited. All rights reserved.